Background

In tandem mass spectrometry, analyte detection is based on collision-induced fragmentation, which is modulated by the collision energy (CE) setting. Variation in CE leads to differential ion yield, and optimization is usually performed empirically as “tuning” during method development. Our aim was to build a method to objectify the impact of collision energy settings on ion yield for individual compounds.

Methods

Collision energy (CE)-breakdown curves were generated based on acquisition files in which a large number of quasi-identical mass transitions were recorded simultaneously, with variation of CE over a defined range within a single injection. Ion yield (normalized to an internal standard recorded with a locked collision energy) was plotted as a curve versus CE settings. Piperacillin and testosterone were studied as exemplary analytes in matrix-free and serum matrix-based liquid chromatography tandem mass spectrometry (LC-MS/MS) measurements. More detailed testosterone CE-breakdown curves were investigated with regard to sample preparation techniques and the isotope labeling pattern of corresponding internal standards.

Results

CE-breakdown curves were found characteristically for the piperacillin quantifier transition with respect to CE-related maximum ion yield, as well as curve width and shape. A diverging curve profile was observed for the piperacillin qualifier transition. For testosterone analyses, no impact from different sample preparation techniques or the isotope labeling patterns on the selected CE was shown.

Conclusion

CE-breakdown curves are a convenient and valuable tool to verify LC-MS/MS methods regarding consistent fragmentation characteristics between sample sources or native analytes and isotope-labeled counterparts.

中文翻译：

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碰撞能量击穿曲线——表征 MS/MS 方法的附加工具

背景

在串联质谱法中，分析物检测基于碰撞诱导的碎裂，该碎裂由碰撞能量 (CE) 设置调节。CE 的变化会导致不同的离子产率，优化通常在方法开发过程中作为“调整”凭经验进行。我们的目标是建立一种方法来客观化碰撞能量设置对单个化合物离子产量的影响。

方法

碰撞能量 (CE) 分解曲线是基于采集文件生成的，在该文件中同时记录了大量准相同的质量跃迁，CE 在单次注射内的定义范围内变化。将离子产率（归一化为使用锁定碰撞能量记录的内标）绘制为曲线与 CE 设置的关系。在无基质和基于血清基质的液相色谱串联质谱 (LC-MS/MS) 测量中研究了哌拉西林和睾酮作为示例性分析物。关于样品制备技术和相应内标的同位素标记模式，研究了更详细的睾酮 CE 分解曲线。

结果

发现了与 CE 相关的最大离子产率以及曲线宽度和形状有关的哌拉西林定量转换的特征性 CE 分解曲线。观察到哌拉西林定性转换的发散曲线。对于睾酮分析，没有显示不同样品制备技术或同位素标记模式对所选 CE 的影响。

结论

CE 分解曲线是一种方便且有价值的工具，可用于验证 LC-MS/MS 方法在样品来源或天然分析物和同位素标记对应物之间的一致碎裂特征。